Hydrogel constructs using stereolithography

ABSTRACT

A preferred embodiment of the present invention provides a method and system for building cost-efficient biocompatible hydrogel constructs using stereolithography. Hydrogel constructs may be used in, for example, multi-lumen nerve regeneration conduits and other tissue engineering scaffolds with embedded channel architecture that facilitate tissue regeneration through possible incorporation of precisely located bioactive agents, cells, and other desired inert and/or active chemical agents and devices. Another preferred embodiment of the present invention provides a method of fabricating a hydrogel construct comprising: solidifying a first solution into a first construct layer with a first energy dosage using stereolithography, the first solution comprising: a first polymer; and a first photoinitiator, wherein the first polymer and first photoinitiator are of a first concentration.

CROSS REFERENCE(S) TO RELATED APPLICATION(S)

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 10/907,984, filed Apr. 22, 2005, now U.S.Pat. No. 7,780,897.

BACKGROUND

The present invention relates to the general field of rapid prototypingtechnology, and in particular, to stereolithography methods and systems.

Rapid prototyping (RP) technologies, also known as Solid FreeformFabrication (SFF), layered manufacturing and other similar technologiesenable the manufacture of complex three-dimensional (3D) parts. RPtechnologies, in particular, generally construct parts by building onelayer at a time. RP technologies are commonly used to build parts andprototypes for use in, for example, the toy, automotive, aircraft andmedical industries. Oftentimes prototypes made by RP technologies aid inresearch and development and provide a low cost alternative totraditional prototyping. In a few cases, RP technologies have been usedin medical applications such as those associated with reconstructivesurgery and tissue engineering (TE).

Stereolithography (SL) is one of the most widely used RP technologiesknown in the art. The resolution of SL machines and the ability of SL tomanufacture highly complex 3D objects, make SL ideal for building bothfunctional and non-functional prototypes. In particular, SL techniquesprovide an economical, physical model of objects quickly and prior tomaking more expensive finished parts. The models are readilycustomizable and changes may be easily implemented.

SL generally involves a multi-stage process. For example, the firststage involves designing and inputting a precise mathematical geometricdescription of the desired structure's shape into one of manycomputer-aided design (CAD) programs and saving the description in thestandard transform language (STL) file format. In the second stage, theSTL file is imported into SL machine-specific software (RP software).The RP software slices the design into layers and determines theplacement of support structures to hold each cross-section in placewhile building the structure layer by layer. By computing buildparameters, the RP software controls the part's fabrication. In thelayer preparation stage, the build parameters for the desired part aretranslated into machine language. Finally, the machine language controlsthe SL machine to build a desired part and its support structure layerby layer. SL machines typically focus an ultraviolet (UV) laser onto across-section of a liquid photopolymer resin. The laser, in turn,selectively cures a resin to form a structure, such as anatomical shapes(i.e., organs and tissues), layer by layer. Ultimately, the part iscleaned, the support structure is removed and the part is post-cured(typically exposed to UV) prior to completion. The operator may,however, need to sand, file or use some other finishing technique on thepart in order to provide a specific surface finish to the structure,which may include painting, plating and/or coating the structure'ssurface.

SL technologies known in the art generally include, for example, alaser, a liquid level sensing system, laser beam optics and controllablescanning mirror system, a vertically movable platform, a single resinretaining receptacle or vat and a recoating device. During the laserscanning phase, a series of optics and controllable scanning mirrorstypically raster a UV laser beam to solidify a photocurable polymerresin. The subject 3D part is first attached to the platform by buildinga support structure with the platform in its topmost position. This stepallows for misalignment between the platform and the surface of theliquid resin—once constructed, the base support structure is parallelwith the surface of the liquid. When building the subject partsimultaneously with its required support structure and after the laserbeam completes a layer, the platform typically is vertically traverseddownward a distance equal to the build layer thickness. After theplatform is vertically traversed downward and prior to selectivelycuring the next layer, a recoating device is typically traversedhorizontally across the part leaving a uniform layer of liquid polymer.The recoating device ensures that trapped spaces within the part arefilled with liquid resin (which may be required for future build layers)and maintains a constant build layer thickness. The process repeats aseach layer is built. Complex-shaped parts are thus manufactured byrepeating the layering process. Once complete, the part is typicallyraised out of the liquid resin, the support structure is removed fromthe part, the part is cleaned and then post-cured. The operator may,however, need to sand, file or use some other finishing technique on thepart in order to provide a specific surface finish to the structure,which may include painting, plating and/or coating the structure'ssurface.

Certain RP technologies facilitate the fabrication of parts used inmedical applications. Such parts require additional designconsiderations. TE techniques, in particular, rely on the use of ascaffold, a framework that provides structural support for cells whilethose cells regenerate the tissue. These scaffolds may also providesignals to the cells to elicit particular desired behaviors. One of themost challenging problems in TE is providing adequate nutrition to cellsseeded within implanted scaffolds. TE techniques known in the art haveshown that the diffusion of oxygen and nutrients is not sufficient tosustain cell viability beyond distances of approximately 75 microns inthe body. Accordingly, TE techniques must retain precise control overthe resulting 3D geometry in order to design favorable diffusion into ascaffold and thus maintain cell viability. Although SL has theresolution and speed to make highly complex 3D structures economically,SL has not been used to aid in TE because SL resins known in the art arenot certified for implantation in humans. Other systems known in the artfor creating complex 3D TE scaffolds are time-consuming and complicatedand therefore are not conducive to mass manufacturing. Accordingly, whatis desired is a system and method of quickly building and mass producingbiocompatible and implantable constructs with precise control overplacement of scaffold materials and bioactive agents and cells topromote favorable tissue regeneration and nutrient diffusion within ascaffold in an economical manner possibly with SL technologies.

Hydrogels are currently being used for a number of different TEapplications, particularly for soft tissues. Hydrogels are biocompatiblematerials with high water content and are suitable as scaffoldingmaterials because of their similarity, both mechanically andstructurally, to extracellular matrices. In addition, hydrogels exhibitfavorable diffusion characteristics and are currently used inphotolithographic processes using manual lithographic masking techniquesas well as a variety of other processes. There are enumerable TEapplications that can benefit from precisely manufacturing hydrogelconstructs with bioactive agents and cells. Hydrogels, however, are notcurrently adequately supported by layered manufacturing (LM)technologies using SL.

Embedded channels may be important to build angiogenic structures orroadways between proliferative structures located within hydrogelscaffolds. Thus, biological and architectural cues need to be assessedto fabricate cytocompatible scaffolds. For example, gradients of growthfactors have been found to direct cell migration and neurite extension,and ultimately enhance tissue regeneration in both guided angiogenesisand subsequent vasculogenesis in vivo and peripheral nerve regeneration.Several agents, such as vascular endothelial growth factor (VEGF), forexample, exert potent angiogenic effects. In the case of VEGF, theseeffects are several-fold, ranging from marrow stimulation of endothelialprecursor production and release to local selective recruitment ofprecursors and enhanced, for example, vascular permeability which inturn enhances vascular bud formation.

Once initiated, a vascular bud is potentially guided by gradients thatallow permeability in the target bud direction and stabilization of theadjacent sides. Several stabilizing agents have been identified invitro. These agents, such as angiopoietin 2, serve to prevent aberrantbudding and to guide the bud in the direction of high permeability. Whenprovided nonspecifically, these agents suppress bud formation. Thus, agradient in VEGF will facilitate guided bud formation and propagation. Areverse gradient of angiopoietin 2 should stabilize directional controlof angiogenesis and prevent nonspecific turns or termination. Further,extracellular matrix (ECM) elements have been shown to either facilitate(hyaluronic acid) or inhibit (polymerized collagen) directionalangiogenesis through specific cellular receptors. Thus, what is desiredis exogenously engineered gradients of biologic agents and/or ECM thatwill potentially facilitate induction and directional propagation ofangiogenesis in an engineered implantable, cytocompatible scaffold.

One particular need in the art is a system and method to create complexnerve guidance conduits. Currently, peripheral nerve repair isaccomplished by using a nerve autograft. Autografting involves taking aportion of a nerve from one location in the body (a donor site) andplacing it in another part of the body exhibiting a specified need.There are several drawbacks to autografting including, for example,requiring multiple surgical sites and a considerable risk of neuromaformation at the donor site. Oftentimes, results from autografting havebeen variable and more often altogether unsuccessful.

Nerve guidance conduits (NGCs) offer a promising alternative toautografting. NGCs are tubes that are sutured to nerve stumps to bridgethe gap and aid in guiding sprouting axons from the regenerating nervetoward their target. NGCs retain neurotrophic factors and othercompounds secreted by the damaged nerve, thus aiding in regeneration andpreventing the infiltration of fibrous tissue. There are currently twotypes of NGCs available, one made of collagen and the other made ofpolyvinylalcohol (a hydrogel). These NGCs, however, are simple, singlematerial and single lumen conduits that fail to recreate the 3Dstructure of the nerve.

Multi-lumen conduits are desirable because they mimic the naturalperipheral nerve structure and increase surface area for neuriteattachment/extension and support cell attachment/migration. Multi-lumenconduits thus allow for more precisely located growth factors andsupport cells within a tissue scaffold. Although multi-lumen conduitsmade with poly(lactic-co-glycolic acid) have previously been made, thetechniques used to make such scaffolds are difficult to scale-up to amanufacturing level, do not allow for cells to be homogeneously seededwithin the conduit during its manufacture, and do not allow themechanical properties of and bioagents within the construct to varythroughout the construct, which is afforded by layered manufacturing.

Thus, systems known in the art fail to mass produce complex, multiplematerial 3D constructs with embedded channel architecture from hydrogelsusing SL technology. Accordingly, what is desired is a low cost,efficient and easy-to-use system which has the ability to fabricatehydrogel constructs with embedded channels of virtually any orientation.What is further desired is a system which enables scaffold fabricationwith internal channel architecture including any variable channelorientation. What is still further desired is the ability to fabricatemultiple material hydrogel constructs that enable the construction ofprecise scaffolds with variable hydrogel scaffold materials both withinand across layers.

In addition, what is still desired is the ability to fabricate multiplematerial hydrogel constructs with precisely placed bioactive agents andcells both within and across layers. What is further desired is a simplemethod for manufacturing multi-lumen conduits of bioactive hydrogels aspotential scaffolds for peripheral nerve regeneration. What is stillfurther desired is a simple method for manufacturing complex bioactivehydrogel constructs as potential scaffolds for guided angiogenesis andadipose tissue generation. What is also still further desired is asimple method for fabricating multi-material constructs that may serveas TE scaffolds.

SUMMARY OF THE INVENTION

One aspect of the present invention overcomes the aforementionedlimitations in an effective and efficient manner, thus expanding the useof RP in various applications and improves SL functionality. In anotheraspect, the present invention will accommodate these needs and providefurther improvements in TE, chemical sensing, biological sensing andnumerous other applications requiring complex, three-dimensional,multi-material, multi-element and/or multi-color, biocompatiblemanufacturing. In still another aspect, the present invention provides amulti-material SL system that builds angiogenic structures or roadwaysbetween proliferative structures for use in, for example, guidedangiogenesis to restore vascular function. In yet another aspect, thepresent invention provides a SL system for constructing bioactive,multi-lumen nerve guidance conduits. In still another aspect, thepresent invention provides a system for fabricating tissue scaffoldssuch as, for example, tissue scaffolds for promoting adipose tissuepopulation and growth.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of the invention may be betterunderstood by referring to the following description in conjunction withthe accompanying drawings, in which:

FIG. 1 is a schematic of a typical SL machine known in the art; and

FIG. 2A depicts the chemical composition of a preferred hydrogel used inan aspect of the present invention;

FIGS. 2B and C depict the preferred chemical compositions preferredphotoinitiators used in an aspect of the present invention;

FIG. 3 illustrates a preferred working environment for building hydrogelconstruct with a typical SL machine (shown with an optional glass slidefor measuring gel thickness);

FIGS. 4A and 4B illustrate the cure depth or hydrogel thickness curvesfor preferred photoinitiators and hydrogel solutions used in an aspectof the present invention;

FIG. 4C illustrates a preferred laser vector pattern used to determinehydrogel thicknesses in accordance with an aspect of the presentinvention;

FIG. 5 illustrates the relationship between gel thickness and energydosage for a preferred solution used in an aspect of the presentinvention;

FIG. 6 depicts a typical UV-VIS absorption spectrum for preferredphotoinitiators used in an aspect of the present invention;

FIGS. 7A-C depict exemplary 3D tissue engineered scaffolds fabricatedwith I-2959 in accordance with an aspect of the present invention;

FIG. 8A depicts the vector file from the SL machine software depictingthe path of the laser beam during the build process in accordance withan aspect of the present invention;

FIGS. 8B and 8C depict an exemplary 3D tissue engineered scaffoldsfabricated with HMPP in accordance with an aspect of the presentinvention;

FIG. 9A depicts a CAD model of a preferred nerve guidance conduit;

FIG. 9B depicts the vector file from the SL machine software depictingthe path of the laser beam during the build process in accordance withan aspect of the present invention; and

FIG. 9C-9F depict exemplary 3D multi-lumen nerve guidance conduitsfabricated in accordance with a preferred embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

A typical prior art SL machine 10, as illustrated in FIG. 1, generallyincludes a UV laser beam 12, a liquid level sensing system 14, optics 16and controllable scanning mirror system 18, a vertically movableplatform 20 and a resin retaining receptacle or vat 22. The vat 22houses, for example, a liquid photocurable polymer resin 24 and,generally, the SL machine 10 rasters a UV laser beam 12 across the resinthrough a series of optics 16 and a controllable scanning mirror system18. In most designs, the subject three-dimensional (3D) part 26 isusually first attached to the platform 20 by building a base supportstructure 28 while the platform 20 is still in its topmost position. Thesupport structure 28 is usually made up of fine filaments that supportthe subject part's 26 overhangs and are manufactured simultaneouslyusing the same resin 24. Prior art designs typically incorporate arecoating device, recoating blade or other sweeping device 29 thatsweeps or horizontally translates across the surface of the liquid afterthe platform 20 and subject part 26 have been traversed downward adistance equal to the build layer thickness. Thus, the recoating device29 facilitates uniform liquid layers on the surface of the subject part26 and eliminates trapped gases or bubbles and/or trapped volumes lefton or underneath the platform 20 and/or the subject part 26 both beforeand during the building process.

Referring still to the prior art SL machine 10 depicted in FIG. 1, afterthe SL machine 10 rasters the UV laser beam 12 and completes a givenlayer (which also includes waiting a sufficient time for the reaction tofinish after the laser beam has completed its scan), the platform 20 isvertically traversed downward a distance equal to the build layerthickness typically between but not limited to 2 and 6 mils oroptionally traversed downward a distance greater than the build layerthickness in order to dip the subject part 26 into the resin 24 and fillany internal part cavities. Once dipping the subject part 26 iscompleted, if dipping is optionally performed, the platform 20 is thentraversed upward until the top of the subject part attached to theplatform is located a distance equal to the build layer thickness fromthe surface of the resin 24. The build layer thickness usually dependson the type of build desired. Prior to beginning a new reaction with thelaser 12, a recoating device 29 typically traverses the liquid resin 24surface as described previously, and the SL machine 10 waits aprescribed amount of time for the liquid resin to reach a state ofequilibrium (so that essentially all waves and any other movement of theliquid resin has stopped) prior to starting the next layer. The processrepeats as each layer is built. The materials used within the layer maybe varied in a number of different configurations including, forexample, the materials used to manufacture the layers can be varied bothwithin and across the layers. Complex-shaped parts are thus manufacturedby repeating the layering process. Once complete, the subject part 26 istypically raised out of the liquid polymer resin 24, the supportstructures 28 are removed and the subject part 26 is cleaned (preferablywith a typical cleaning solution or a cytocompatible solution) andpost-cured, usually in a UV oven (not shown). However, it should beunderstood that support structures 28 may be removed before, during,and/or after the cleaning and curing/drying processes.

In order to attach newly formed layers to previously cured layers, it iscrucial to maintain a certain liquid photocurable resin 24 chemistry,layer thickness and laser energy so that the laser 12 is capable ofcuring beyond the layer thickness into previously cured layers (alsoknown as “laser overcure”). Cure depth, a fundamental characteristic ofliquid photocurable resins 24, measures the penetration depth of a laserat which the laser successfully cures the liquid photocurable resin 24for a given laser energy. As described earlier, in a typical commercialSL machine 10 a series of optics 16 focuses and directs the laser 12onto the liquid surface while the scanning mirrors 18 controls thelaser's movement in the X-Y plane across the vat 22. Because commercialSL machines 10 accommodate a dynamic scanning mirror system,photochemical reactions are controlled by varying the scan speed of thelaser 12 (for a fixed laser power). Thus, understanding andcharacterizing the cure depth behavior of the liquid photocurable resins24 is necessary for successfully fabricating 3D objects using SL and itsscanning mirror system 18 as seen, for example, in the later-describedexample.

Additionally, in order to successfully fabricate embedded channels andfeatures that are opened or closed in and/or between cured layers, forexample, the particular characteristics of the hydrogel solution (e.g.,a photoinitiator (PI), poly(ethylene glycol) (PEG) and distilled watersolution) need to be characterized. Certain unique characteristics mustbe exhibited in order to allow for successful fabrication. For example,some hydrogel solutions exhibit characteristics, such as hydrogelthickness or cure depth, that may potentially vary widely as a functionof PI type and concentration, energy dosage and polymer concentration inthe hydrogel solution as also demonstrated in the later-describedexample.

Polymers that may be used in the present invention include any number ofpolymers with photoreactive functional groups and are capable of forminga hydrogel construct. These polymers include natural, synthetic, andsemi-synthetic polymers known to those of skill in the art, and may bedegradable or non-degradable. One exemplary polymer is derivatized PEGhaving functional groups such as acrylate, methacrylate, or vinylsulfone. The PEG may have a molecular weight between about 1000 and20,000, preferably between about 1000 and 10,000. The PEG may be astraight linear chain with a functional group on either end, such as PEGdiacrylate, or may be a multi-armed PEG. Other polymers which may beused include, for example, derivatized polyvinyl alcohol, hyaluronicacid, chondroitin sulfate, and collagen. For example, methacrylatedhyaluronic acid and methacrylated chondroitin sulfate have both beenphotocrosslinked into hydrogels. Other useful polymers capable offorming hydrogels following photocrosslinking are well-known to those ofskill in the art. The concentration of the polymer in solution ispreferably between about 1% and 30% (w/v).

Various biocompatible fluids may be used in conjunction with aspects ofthe present invention, including water, buffer (such asphosphate-buffered saline) and cell culture media. When cells orbioactive factors are included in the polymer solution, the fluid ispreferably at a pH of about 7.0 to 7.8, most preferably about 7.4.

Various PIs may be used in conjunction with a preferred embodiment ofthe present invention and are generally known to those of skill in theart of photocrosslinking. The PI preferably has an absorption in the UVwavelength range, and more preferably in the long UV wavelength range.Preferred PIs, include but are not limited to,2,2-dimethyl-2-phenylacetophenone,2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP), and2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959, Ciba-Geigy). The concentration of the PI in the polymer solutionis preferably below about 5%, and more preferably below about 1%,particularly when cells are present in the polymer solution. It shouldbe understood by those skilled in the art that other PIs, such as otherUV-PIs and visible PIs, may also be used in accordance with aspects ofthe present invention.

Bioactive factors, such as adhesion ligands, growth factors, andcytokines, may be incorporated into the scaffold during thephotocrosslinking process by including them in the polymer solution.These bioactive factors may be attached to the scaffold during thephotocrosslinking process through functional groups attached to thefactors, or may simply be trapped within the hydrogel during theprocess. The bioactive factors are preferably incorporated at aconcentration effective to elicit the desired biological response.

Cells may be included in the polymer solution prior to thephotocrosslinking process, thereby being homogeneously seeded within thescaffold following photocrosslinking of the hydrogel (or seeded into thescaffold after construction). The cell density may be wide-rangingdepending on the particular application for which the scaffold will beused, but will typically not be more than about 50×10⁶ cells/ml in thepolymer solution.

In accordance with one aspect of the present invention, a complete TEcytocompatible hydrogel implant or construct was fabricated by firstcreating a desired nerve regeneration conduit computer-aided design(CAD) drawing. The design is saved in a standard transform language(STL) file format and imported in SL machine-specific software to createSL vector files or SL machine command files. Contemporaneously, hydrogeland PI selection occurs depending on the desired design criteria. Asolution is prepared combining the hydrogel in distilled water and PI.Optionally, the solution may incorporate additives including, forexample, bioactive agents, live cells, other chemicals, devices forimplantation or any combination thereof. The solution may undergoseveral characterization tests including, for example, tests forevaluating mechanical, photo-chemical and cytocompatibility properties.After the initial design and testing phases are complete, the SL processmay begin. After initialization of the SL machine, including laser poweradjustment, SL machine-specific parameter determination, the hydrogelreceptacle (or other existing receptacle), is placed at a desired heighton the building platform. After determining the receptacle and solutionvolume required for a desired build layer thickness, the build processbegins. Keeping in mind that build layer thickness may be varied as thebuild process continues, certain material volumes and machine parametersmay need to be altered.

For example, to begin the building phase, an initial amount of solutionis added into the receptacle, either by hand or by way of an automatedsystem free of human intervention (or alternatively, the solution iscontained in a fixed material vat similar to those found in existingcommercial systems). Part building begins in accordance with the layerspecific build file or machine command file using a machine adapted toperform SL including, for example, the SL machine schematically depictedin FIG. 3 without the optional glass slide. As each hydrogel constructlayer is complete, the platform is traversed vertically a distance equalto the desired build layer thickness (which can be varied throughout thebuild).

In accordance with a preferred embodiment of the present invention, eachhydrogel construct layer (or portion of a layer) may be fabricated toreflect a certain characteristic. As mentioned earlier, uniquecharacteristics, such as hydrogel thickness or cure depth, vary widelyas a function of PI type and concentration, energy dosage and polymerconcentration in the hydrogel solution as also demonstrated in thelater-described example. Thus, by changing PI type, PI concentration,polymer type, polymer concentration, solution type or solutionconcentration or energy dosage, the hydrogel solution generally exhibitsa unique characteristic. Accordingly, in a preferred embodiment,although the entire hydrogel construct may be made of essentially thesame material, distinct construct layers (or a portion of a layer), forexample, exhibit unique physical and biological characteristicsdepending on the altered characteristics of the hydrogel solution usedto fabricate that particular construct layer (or portion of a layer).Alternatively, it may be desired to build a layer (or a portion of apart layer) within or across the current part layer with altogetherdifferent materials. Keeping in mind that each layer may be built in anumber of different configurations including, for example, partiallayers, layers built in any one dimension or in multiple dimensions andcross-layers built between layers in any dimensions, a preferredembodiment of the present invention provides a system of fabricatingvirtually endless combinations of single material and multi-materialhydrogel constructs, allowing different parts of the same hydrogelconstruct to exhibit a desired characteristic.

As each part layer is completed, solution is added or removedaccordingly to or from, respectively, from the receptacle to accommodatevariable build layers (or portions of a build layer) and layer thickness(or thickness of a portion of a layer), using a receptacle fill/removesystem (or alternatively, the platform could be traversed down into asingle vat of material). The preferred building process is repeatedusing the build or machine command file until the part is complete.Finally, after removing all of the solution, the subject part isseparated from the receptacle (or, alternatively the subject part isseparated from an existing build platform or from an optional buildplatform such as a glass slide as seen in FIG. 3), the subject part iscleaned, optionally undergoes a finishing process that may includecutting or trimming the hydrogel construct, rinsing with acytocompatible solution, and finally inspected for quality control.Accordingly, a completed TE hydrogel implant is cytocompatible andfabricated using SL. TE hydrogel implants additionally may promote, forexample, adipose tissue population and growth (or nerve regeneration orguided angiogenesis and ultimately vasculogenesis).

Although the below-described example primarily references PEG, it shouldbe understood by those skilled in the art that other hydrogels may alsobe used in accordance with the present invention. For example, a naturalpolymer, synthetic polymer or some combination thereof may also be used.Natural polymer hydrogels include polymers such as anionic polymers (forexample, hyaluronic acid, alginic acid, pectin, carrageenan, chondroitinsulfate, dextran sulfate), cationic polymers (for example, chitosan andpolylysine), amphipathic polymers (such as collagen, gelatin,carboxymethyl chitin and fibrin) and neutral polymers (for example,dextran, agarose and pullulan) and their derivatives.

Synthetic polymer hydrogels, on the other hand, include, for example,polymers such as polyesters: poly(ethylene glycol)-poly(lacticacid)-poly(ethylene glycol); poly(ethyleneglycol)-poly(lactic-co-glycolic acid)-poly(ethylene glycol);poly(ethylene glycol)-polycaprolactone-poly(ethylene glycol);poly(lactic acid)-poly(ethylene glycol) -poly(lactic acid);poly(hydroxyl butyrate); poly(propylene fumerate-co-ethyleneglycol)±acrylate end groups; and poly(poly(ethyleneglycol)/poly(butylene oxide)terephthalate).

Synthetic polymer hydrogels may include, for example, other polymerssuch as: poly(ethylene glycol)-bis-(poly(lactic acid)-acrylate);poly(ethylene glycol)±cyclodextrins; poly(ethyleneglycol)-g-poly(acrylamide-co-Vamine); polyacrylamide; poly(N-isopropylacrylamide-co-acrylic acid); poly(N-isopropyl acrylamide-co-ethylmethacrylate); poly(vinyl acetate)/poly(vinyl alcohol); poly(N-vinylpyrrolidone); poly(methyl methacrylate-co -hydroxyethyl methacrylate);polyacrylonitrile-co-allyl sulfonate);poly(biscarboxy-phenoxy-phosphazene); and poly(glucosylethylmethacrylate-sulfate).

Combinations of natural and synthetic polymer hydrogels may includepolymers such as poly(polyethylene glycol-co-peptides), alginateg-(polyethylene oxide-polypropylene oxide-polyethylene oxide),poly(polylactic-co-glycolic acid-co-serine), collagen-acrylate,alginate-acrylate, poly(hydroxyethly methacyrlate-g-peptide),poly(hydroxyethyl methacyrlate/Matrigel®) and hyraluronicacid-g-N-isopropyl acrylamide).

The SL materials used in accordance with a preferred embodiment of thepresent invention may be rigid, semi-rigid, liquid (may be encapsulatedliquid) or gas (trapped gases). There are numerous examples of curablefluid media 24 suitable for use with aspects of the present invention.Examples of curable fluid media 24 or materials that may be incorporatedinto the curable fluid media 24 include SL resins known in the art,hydrogels, bioactive ingredients, cells, imbedded devices or materials,photopolymer resins and powdered materials. Some types of powderedmaterials may be converted from a fluid-like medium to a cohesivecross-section by processes, such as melting and solidification.

In addition, in accordance with a preferred embodiment of the presentinvention, multi-colored manufacturing may be accomplished by mixingpigments, fluorescing particles, paints, dyes and/or other color mediainto the curable fluid medium 24, thereby facilitating the manufactureof multi-colored prototypes and models. Similarly, other materials may,optionally, be mixed into the fluid medium 24 to alter the strength,thermal, mechanical, optical, electrical, functional and/orbiofunctional properties thereby facilitating the manufacture ofmulti-functional, multi-material, multi-colored, multi-element and/orimplantable prototypes, models and finished products. The presentinvention thus facilitates using SL technology to aid in manufacturingof parts in an endless number of materials and colors. The presentinvention also facilitates manipulating certain materials to exhibitaltered properties at select locations during the building stage.

EXAMPLE

Poly(ethylene glycol) (PEG) is an example of a synthetic hydrogelmaterial which is cytocompatible and potentially has important uses intissue regeneration. PEG is generally non-toxic, non-immunogenic and canbe easily cleared from the body. In addition, PEG is water soluble andcan be easily modified with photoreactive and crosslinkable groups likeacrylates or methacrylates. Thus, PEG is ideal for creatingphotocrosslinkable hydrogel tissue scaffolds. The systems and methods inaccordance with an aspect of the present invention make possible, forexample, successful fabrication of 3D PEG-based scaffolds using SL.

Generally, it was found that hydrogel thicknesses vary at select energydosages for different scan speeds of the SL machine's UV scanningsystem. In fact, hydrogel thickness was found to be a strong function ofPI type and concentration, energy dosage and PEG-dimethacrylate(PEG-dma) concentration (for a molecular weight, M_(w), of 1000 PEG-dmacommercially available), especially at the low PI concentrationsrequired for implantation. Hydrogel thickness curves demonstrate LM fortwo construct geometries where different layer thicknesses were requiredto successfully fabricate the constructs. Thus, an aspect of the presentinvention demonstrates, for example, the effective use of SL as aprocessing technique for complex cytocompatible 3D tissue scaffolds. Inaddition, other aspects of the present invention address, for example,practical considerations associated with the use of hydrogels in LM.

In accordance with an aspect of the present invention, PEG-dma M_(w)1000 was used to prepare two solutions with different concentrations(20% and 30% w/v) in distilled water. Aliquot portions were separatedfrom these solutions, and different concentrations of two PIs were addedto the portions. The PIs used in this instance were Sarcure 1121 or HMPP(2-hydroxy-2-methyl-1-phenyl-1-propanone) and Irgacure 2959 or I-2959(2-hydroxy-1-[4-(2-hydroxehtoxy)phenyl]-2-methyl-1-propanone). Thechemical structures of the PEG-dma and the two PIs used in this exampleare shown in FIGS. 2A, 2B and 2C, respectively.

In order to obtain viable hydrogel thickness curves, the movableplatform 20 of the SL machine 10 was set a fixed height and fitted witha laser 12, specifically a He—Cd laser (325 nm) as seen in, for example,FIG. 3. The PEG-dma solution with PI was pipetted inside a flat-topcylindrical container 30 and filled to the rim. A glass slide 32 wasplaced on top of the container 30 and in contact with the PEG-dmasolution. The glass slide 32 acts as a substrate for hydrogel attachmentand facilitates the thickness measurements. It was determined that theglass slide 32 filters approximately 18% of the laser 12 power.

The cylindrical container 30 with the glass slide 32 was placed on thecenter of the platform 20 as depicted in FIG. 3. At the test height ofthe platform 20, the laser 12 was circular with a diameter ofapproximately 250-micrometers. The samples were cured by writing avector pattern through the glass slide 32 and into the PEG-dma container30 at different energy doses. FIG. 4C, for example, illustrates thevector file of the actual laser beam trace on laser burn paper. Apreferred laser vector pattern 36 used to determine hydrogel thicknessesin accordance with an aspect of the present invention. The pattern 36,in this example, consisted of a series of nineteen parallel linesapproximately 250 micrometers wide (the laser beam diameter) and 7.62 mmlong, spaced approximately 355-micrometers apart.

The laser 12 drew each line in the pattern 36 twice and the energydosage was varied by changing the SL machine 10 parameters that controlthe scan speed of the laser 12. After polymerization, the glass slide 32was lifted off of the container 34 with the polymerized hydrogelattached to the slide. The cured hydrogels were rinsed with distilledwater to remove any unreacted polymer and then measured with, forexample, a caliper. This procedure was repeated for all hydrogelthickness measurements. Four sample hydrogels were cured for the PEG-dmasolutions with I-2959, and two sample gels were cured for the solutionswith HMPP. It should be noted that the measured power of the He—Cd laser12 (rated at 40 mW) at the fixed platform 20 height was 29.5 to 30.6 mWand 35.8 to 37.1 mW with and without the glass slide 32, respectively.The measured laser 12 powers with the glass slide 32 were used todetermine the laser energies.

The resulting hydrogel thickness curves as a function of PI type andconcentration and the energy dose are shown in FIGS. 4A and 4B, whereFIG. 4A represents the gel thickness curve when I-2959 is used as a PIand FIG. 4B represents the same for HMPP. The three markers used inFIGS. 4A and 4B correspond to three different energy dosages. Thediamond shape (♦) represents an energy level of 3.604 j/cm² or 0.258IPS. The square shape (▪) represents an energy level of 1.640 j/cm² or0.567 IPS. The triangle shape (▴) represents an energy level of 0.586j/cm² or 1.585 IPS.

FIGS. 4A and 4B also illustrate that there are significantly greater gelthicknesses achieved with HMPP when compared with I-2959 for a given PIconcentration and energy dosage. It was further observed that higherpolymer concentrations typically produce thicker hydrogels. Similarly,higher energy dosages generally produce thicker gels. Thus, the hydrogelthickness curves aid in prescribing layer thicknesses for fabricatingcomplex 3D scaffolds. For example, the I-2959 affords polymerization ofthin layers and therefore the fabrication in a layer-by layer fashion.On the other hand, HMPP may be used to successfully fabricate singlelayer “large” structures.

FIG. 5 illustrates that there is a relationship between energy dosageand hydrogel thickness for PEG solutions with 0.5% (w/v) I-2959. Thesolid diamond markers (♦) correspond to 0.5% (w/v) of I-2959, while thehollow diamond markers (⋄) correspond to 20% (w/v) PEG-dma in distilledwater. Again, as seen in FIG. 5, for the two types of PIs tested, at lowPI concentrations (<0.05%) and small energy dosages (fast scanningspeeds) the hydrogels produced were thick and loosely crosslinked.

FIG. 6 illustrates the UV-VIS absorption spectrum of the two PIs used inthe gel thickness experiments and shows, for example, that the hydrogelthickness has a maximum at low PI concentrations and decreasesasymptotically to a non-zero value as PI concentrations increase.Hydrogel thickness generally begins at zero for zero PI concentration,has a maximum at low PI concentrations, and decreases asymptotically toa non-zero value as PI concentration increases. Hydrogel thicknessstarts at zero due to the presence of polymerization inhibitors,including monomethyl ether hydroquinone (MEHQ) and butylatedhydroxytoluene (BHT), added by the manufacturer in the PI. Measurementin the region between zero and the maximum gel thickness are not presenthere as the gel is loosely crosslinked at these PI concentrations andthe measurements are highly uncertain (and thus, the maximum gelthickness presented here should not be viewed as an absolute maximum).

As mentioned earlier, I-2959 affords polymerization of thin layers andtherefore the fabrication in a layer-by layer fashion, while HMPP may beused to successfully fabricate single layer “large” structures.Accordingly, a preferred embodiment of the present invention provides SLprocessing to fabricate cytocompatible parts such as those seen in FIGS.7, 8 and 9. For example, FIG. 7A depicts the complex 3D structure withembedded 3D channel architecture encoded in a vector file. The vectorfile was in turn used to fabricate the scaffold depicted in FIGS. 7B and7C using I-2959. As another example, FIG. 8A depicts the relativelysimple structure with multiple straight channels encoded in a vectorfile. This vector file was used to fabricate the scaffold in one layerusing HMPP as in FIGS. 8B and 8C.

FIGS. 9A-9F depict other examples of multi-lumen and multi-layered nerveguidance conduits which may be built in accordance with one aspect ofthe present invention. FIG. 9A depicts a CAD model of a preferred nerveguidance conduit while FIG. 9B depicts the vector file from the SLmachine software depicting the path of the laser beam during the buildprocess in accordance with an aspect of the present invention; and FIG.9C-9F depict exemplary 3D multi-lumen nerve guidance conduits fabricatedin accordance with a preferred embodiment of the present invention.

It should be understood by those skilled in the art that there arenumerous other shapes, sizes and configurations of tissue scaffolds andnerve guide conduits, for example, which may be fabricated using apreferred embodiment of the present invention. Although preferredembodiments of the present invention have been described in detail, itwill be understood by those skilled in the art that variousmodifications can be made therein without departing from the spirit andscope of the invention as set forth in the appended claims.

1. A method of fabricating a hydrogel construct comprising: solidifyingone or more solutions into three or more hydrogel construct layers suchthat: (i) the plurality of hydrogel construct layers are coupledtogether to form the construct; and (ii) at least one lumen or channelextends through at least two of the three or more hydrogel constructlayers; where each of the one or more solutions comprise a hydrogelpolymer and a photoiniator.
 2. The method of claim 1, where the three ormore hydrogel construct layers are solidified such that two or morelumens or channels extend through at least two of the three or morehydrogel construct layers.
 3. The method of claim 2, where at least twoof the two or more lumens or channels connect within the construct. 4.The method of claim 1, where at least one of the three or more layers issolidified from two or more different solutions.
 5. The method of claim1, where at least one of the hydrogel polymer(s) is a derivative ofpoly(ethylene glycol).
 6. The method of claim 3, where at least one ofthe photoinitiator(s) is selected from the group consisting of UVphotoinitiators and visible-light photoinitiators.
 7. The method ofclaim 1, where at least one of the photoinitiator(s) is selected fromthe group consisting of: 2-hydroxy-2-methyl-1 phenyl-1-propanone,2-hydroxy-1-[4-(2-hydroxehtoxy)phenyl]-2-methyl-1-propanone,2,2-dimethoxy-2-phenyl-acetophenone, and any combination thereof.
 8. Themethod of claim 1, where at least one of the one or more solutionsincludes one or more materials selected from the group consisting of:biocompatible media, water, a buffer, phosphate-buffered saline, cellculture media, and any combination thereof.
 9. The method of claim 1,where the construct is cytocompatible.
 10. The method of claim 3, whereat least one of the hydrogel construct layers has a thickness that isdifferent from the thickness of at least one other of the three or morehydrogel construct layers.
 11. The method of claim 3, where at least oneof the three or more hydrogel construct layers is a partial layer. 12.The method of claim 1, further comprising cleaning the hydrogelconstruct.
 13. The method of claim 1, further comprising curing/dryingthe hydrogel construct.
 14. The method of claim 1, where at least one ofthe one or more solutions includes an additive selected from the groupconsisting of imbedded devices and imbedded materials, bioactiveingredients and cells, and any combination thereof.
 15. The method ofclaim 1, where the one or more solutions are solidified usingstereolithography.